This proposal is for the second competitive renewal of grant HL62969, focusing on the biophysics of nitrosyl heme chemistry and its role in nitric oxide (NO) signaling. NO is a reactive molecule with a short half-life in biological solutions and many of the reaction products are toxic. Yet, NO is produced in all mammalian tissues and is central to a wide-ranging array of physiological processes, including vasodilation, blood coagulation, memory formation, inflammation, angiogenesis, cell growth and cell death. The reactive nature of NO and its pervasive physiological nature have made NO signaling pathways attractive targets for intervention in many diseases, including cardiovascular disease, cancer, diabetes and asthma. In past funding period, we uncovered the means by which the nitrophorins, NO transport proteins from blood-sucking insects, are able to overcome the reactive nature of NO and successfully transport and release the molecule while obtaining a blood meal. We also developed expression systems for full-length and functional fragments of soluble guanylyl cyclase (sGC), which plays a key role in transducing the NO signal, and developed a model cell system in which to examine sGC activity under the influence of the cellular environment. For the next funding period, we have three specific aims. Specific Aim 1 is to obtain atomic models of sGC and other NO-sensitive heme proteins, including the nitrophorins. Experiments involving X-ray crystallography, electron microscopy and mutagenesis are proposed. Specific Aim 2 is to understand mechanism in the allosteric regulation of sGC. Experiments involving ligand binding, kinetics, femtosecond photolysis and 2D-IR are proposed. Specific Aim 3 is to understand regulation of sGC in the cell, using a human fibrosarcoma cell line that we have engineered to have NO synthase under Tet regulation, and sGC with purification tags. These cells respond to external factors that regulate sGC activity and cell motility, factors of importance in blood pressure regulation, angiogenesis and metastasis. We will use proteomic, molecular biology and cellular imaging approaches to uncover regulatory modifications, protein-protein interactions and cellular distribution. We are conducting molecular studies on nitric oxide signaling pathways that control blood pressure, cell migration and many other physiological processes. Our results are expected to uncover new approaches for drug discovery targeted to cardiovascular disease, cancer metastasis and related failures in human health.